Oligonucleotide sequences that identify species of animal

ABSTRACT

The present invention provides a method for identifying animal species, said method comprises a step of amplifying a DNA fragment by PCR using a DNA in a sample as a template and animal-specific DNA sequences as a primer pair, wherein the animal-specific DNA sequences are derived from a ATP synthase subunit 8 gene or a region proximal thereto of a mitochondrial genome; and a step of detecting the amplified DNA fragment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 13/417,692filed Mar. 12, 2012, which is a Division of application Ser. No.12/707,828 filed Feb. 18, 2010, which is a Continuation of applicationSer. No. 10/826,119 filed Apr. 16, 2004, the entire contents of all ofwhich is hereby incorporated by reference. Applicants request use of thecompliant computer readable “Sequence Listing” that is already on filefor application Ser. No. 10/826,119 filed Apr. 16, 2004 and state thatthe paper or compact disc copy of the “Sequence Listing” in thisapplication is identical to the computer readable copy filed forapplication Ser. No. 10/826,119 filed Apr. 16, 2004.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for identifying animal speciesand animal-derived DNA-specific primer pairs used therein. Morespecifically, the present invention relates to methods for identifyinganimal species that include a step of amplifying an animal-specific DNAsequence derived from the ATP synthase subunit 8 gene of a mitochondrialgenome, and primer pairs used in this amplification step.

2. Description of the Related Art

Currently, there is a problem that cattle is infected with bovinespongiform encephalopathy (BSE) by giving feed containing meat andbonemeal derived from cattle infected with BSE. It has been shown thatBSE-like diseases may be present in various livestock as well.Accordingly, since the emergence of BSE, there has been a need todevelop sensitive and effective method to identify feed contaminatedbone and bonemeal, and this has become a particularly urgent matter forauthorities.

Immunological methods and gene identification methods using nuclear geneconventionally have been used as methods for identifying animal species.Examples of immunological methods include ELISA and immunoblotting. PCRis an example of a gene identification method using nuclear gene.However, there are many problems with methods for identifying the animalspecies that are currently employed. For example, in meat and bonemealthat has been heat-treated, there is a high likelihood that nucleicacids have been fragmented. Furthermore, majority of feed in which meatand bonemeal has been mixed is composed of plant-derived material.Therefore, it is necessary to analyze trace amounts of animal-derivedcomponents. There is a dire need for the development of a detectionmethod that is highly sensitive and effective and that can be executedwith respect to such heat-treated samples.

Accordingly, a method other than immunological methods and geneidentification methods employing nuclear gene that is for detecting andidentifying the animal species of animal-derived components that arepresent in trace amounts is desirable. In particular, it is crucial toidentify the type of animal meat, meat and bonemeal, or fishmeal used infeed given to livestock and pets. Furthermore, it is desirable that thedetection method is highly sensitive and differentiate species of animalDNA present in trace amounts from among large quantities of plant DNA orDNA of other animal species.

SUMMARY OF THE INVENTION

The inventors of the present application focused on the mitochondrialgenome, which is inherited maternally and exists in a greater number ofcopies than nuclear genome, and investigated the use of mitochondrialgene as a target for identifying animal species. Their researchindicated that the homologous sequence with ATP synthase subunit 8 gene(atp8 gene) from animal mitochondrial genome is not present in plant(Oryza sativa) mitochondrial genome. Thus, the inventors found that theDNA derived from the atp8 gene can serve as a material for specificdetection of trace amounts of animal DNA among plant-based feed, inother word, plant atp8 gene is very diverged from animal atp8 gene, andalso that specific DNA sequences of the animal mitochondrial atp8 genecan be used to identify the animal species, thereby arriving at thepresent invention.

The present invention provides a method for identifying animal species,said method comprises:

amplifying a DNA sequence by PCR using a DNA in a sample as a templateand animal-specific DNA sequences as a primer pair, wherein theanimal-specific DNA sequences are derived from a ATP synthase subunit 8gene or a region proximal thereto of a mitochondrial genome, and

detecting the amplified DNA sequence.

In a preferred embodiment, the animal is a mammal, and furtherpreferably, the primer pair is a combination of the DNA sequence of SEQID NO: 1 and the DNA sequence of SEQ ID NO: 2.

In a further preferred embodiment, the mammal is selected from the groupconsisting of cattle, sheep, goat, deer, pig, horse, rabbit, and whale.

In a preferred embodiment, the animal is a ruminant, and furtherpreferably, the primer pair is a combination of the DNA sequence of SEQID NO: 3 and the DNA sequence of SEQ ID NO: 4, or a combination of theDNA sequence of SEQ ID NO: 5 and the DNA sequence of SEQ ID NO: 6.

In a further preferred embodiment, the ruminant is selected from thegroup consisting of cattle, sheep, goat, and deer.

In a preferred embodiment, the animal is a cattle, and furtherpreferably, the primer pair is a combination of DNA sequences selectedfrom the group consisting of the following DNA sequence combinations:SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 12; SEQ IDNO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 12; SEQ ID NO: 11and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12; and SEQ ID NO: 14 andSEQ ID NO: 15.

In a preferred embodiment, the animal is a pig, and further preferably,the primer pair is a combination of the DNA sequence of SEQ ID NO: 17and the DNA sequence of SEQ ID NO: 19, or a combination of the DNAsequence of SEQ ID NO: 18 and the DNA sequence of SEQ ID NO: 22.

In a preferred embodiment, the animal is a sheep, and furtherpreferably, the primer pair is a combination of the DNA sequence of SEQID NO: 23 and the DNA sequence of SEQ ID NO: 24.

In a preferred embodiment, the animal is a goat, and further preferably,the primer pair is a combination of the DNA sequence of SEQ ID NO: 25and the DNA sequence of SEQ ID NO: 26.

In a preferred embodiment, the animal is a chicken, and furtherpreferably, the primer pair is a combination of the DNA sequence of SEQID NO: 28 and the DNA sequence of SEQ ID NO: 30.

In a preferred embodiment, the animal is a fish, and further preferably,the primer pair is a combination of the DNA sequence selected from thegroup consisting of SEQ ID NOS: 32, 34, 38 and 39 and the DNA sequenceselected from the group consisting of SEQ ID NOS: 33, 35, 36, 37, 40,and 41.

In a further preferred embodiment, the fish is selected from the groupconsisting of sardine, flatfish, salmon, Alaska Pollack, tuna, and ladycrab.

In a further preferred embodiment, the sample is selected from a groupconsisting of raw meat, raw fish, processed meat food products,processed fish food products, food products containing processed meat,food products containing processed fish, blood, hair, body fluids, milk,milk processing products, meat and bonemeal, bonemeal, fishmeal, fishsoluble, and feed, fertilizer, and feed additive containing them.

The present invention also provides a primer pair for detection of amammal-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 1 and the DNA sequence of SEQ ID NO: 2.

In a preferred embodiment, the mammal is selected from the groupconsisting of cattle, sheep, goat, deer, pig, horse, rabbit, and whale.

The present invention also provides a primer pair for detection of aruminant-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 3 and the DNA sequence of SEQ ID NO: 4, or acombination of the DNA sequence of SEQ ID NO: 5 and the DNA sequence ofSEQ ID NO: 6.

In a preferred embodiment, the ruminant is selected from the groupconsisting of cattle, sheep, goat, and deer.

The present invention also provides a primer pair for detection of acattle-specific DNA, the primer pair being a combination of DNAsequences selected from the group consisting of the following DNAsequence combinations: SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 andSEQ ID NO: 12; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ IDNO: 12; SEQ ID NO: 11 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 12;and SEQ ID NO: 14 and SEQ ID NO: 15.

The present invention also provides a primer pair for detection of apig-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 17 and the DNA sequence of SEQ ID NO: 19, or acombination the DNA sequence of SEQ ID NO: 18 and the DNA sequence ofSEQ ID NO: 22.

The present invention also provides a primer pair for detection of asheep-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 23 and the DNA sequence of SEQ ID NO: 24.

The present invention also provides a primer pair for detection of agoat-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 25 and the DNA sequence of SEQ ID NO: 26.

The present invention also provides a primer pair for detection of achicken-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 28 and the DNA sequence of SEQ ID NO: 30.

The present invention also provides a primer pair for detection of afish-specific DNA, the primer pair being a combination of the DNAsequence selected from the group consisting of SEQ ID NOS: 32, 34, 38and 39 and the DNA sequence selected from the group consisting of SEQ IDNOS: 33, 35, 36, 37, 40, and 41.

In a preferred embodiment, the fish is selected from the groupconsisting of sardine, flatfish, salmon, Alaska Pollack, tuna, and ladycrab.

The present invention also provides a primer pair for detection of aplant-specific DNA, the primer pair being a combination of the DNAsequence of SEQ ID NO: 42 and the DNA sequence of SEQ ID NO: 43.

Further, the present invention provides a method for detectinganimal-derived components present in mixed feed, said method comprises:

amplifying a DNA sequence by PCR using a DNA in a sample as a templateand animal-specific DNA sequences as a primer pair, wherein theanimal-specific DNA sequences are derived from a ATP synthase subunit 8gene or a region proximal thereto of a mitochondrial genome, and

detecting the amplified DNA sequence.

The present invention also provides a kit for detecting ananimal-derived component present in a sample, said kit comprises atleast one of primer pair specific for any animal described above.

In a preferred embodiment, the kit further comprises a primer pair fordetection of a plant-specific DNA, said primer pair is a combination ofthe DNA sequence of SEQ ID NO: 42 and the DNA sequence of SEQ ID NO: 43.

Further, the present invention provides a method for detectingplant-derived components present in sample, said method comprises:

amplifying a DNA sequence by PCR using a DNA in a sample as a templateand plant-specific DNA sequences as a primer pair, wherein theplant-specific DNA sequences are derived from a ATP synthase subunit 8gene or a region proximal thereto of a mitochondrial genome, and

detecting the amplified DNA sequence.

Using the method of the present invention, it is possible to detect,with high sensitivity, trace amounts of animal-derived DNA in a sample.It is thus applicable to identify the animal species of trace amounts ofmeat and bonemeal mixed into feed. More particularly, detection ispossible even if trace amounts of cattle-derived meat and bonemeal aremixed into mixed feed for livestock. Further, the primers for thedetection of fish-specific DNA according to the present invention areuseful for detecting a wide range of fishmeal derived from various fishspecies that has been mixed into mixed feed or the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a sequence alignment of mitochondrial atp8 genes derivedfrom various types of animals.

FIG. 2 shows a sequence alignment of chicken, cattle, and pigmitochondrial atp8 genes.

FIG. 3 shows a sequence alignment of mitochondrial atp8 genes, andnearby regions, derived from various types of animals.

FIG. 4 shows the positional relationship on the mitochondrial atp8 geneof the primers used to specifically detect cattle-derived DNA sequences.

FIG. 5 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (anicon5 and anicon3) for detectingmammal-specific DNA sequences and with DNAs derived from various typesof animals serving as templates.

FIG. 6 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (rumicon5 and rumicon3) for detectingruminant-specific DNA sequences and with DNAs derived from various typesof animals serving as templates.

FIG. 7 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (Fpr-F and Fpr-R) for detectingruminant-specific DNA sequences and with DNAs derived from various typesof animals serving as templates.

FIG. 8 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (cow3 and cow51) for detectingcattle-specific DNA sequences and with DNAs derived from various typesof animals serving as templates.

FIG. 9 is a photograph showing the result of electrophoresis afterperforming PCR using various primer pairs for detecting cattle-specificDNA sequences and with DNAs derived from various types of animalsserving as templates.

FIG. 10 is a photograph showing the results of electrophoresis afterperforming PCR using various primer pairs for detecting cattle-specificDNA sequences and with DNAs derived from various types of animalsserving as templates.

FIG. 11 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (Fpc-F and Fpc-R) for detectingcattle-specific DNA sequences and with DNAs derived from various typesof animals serving as templates.

FIG. 12 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (pig51 and pig3) for detectingpig-specific DNA sequences and with DNAs derived from various types ofanimals serving as templates.

FIG. 13 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (pig5-3 and pig32-2) for detectingpig-specific DNA sequences and with DNAs derived from various types ofanimals serving as templates.

FIG. 14 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair (sheep5 and sheep3) for detectingsheep-specific DNA sequences and a primer pair (goat5 and goat3) fordetecting goat-specific DNA sequences, and with DNAs derived fromvarious types of animals serving as templates.

FIG. 15 is a photograph showing the results of electrophoresis afterperforming PCR using a primer pair for detecting chicken-specific DNAsequences and with DNAs derived from various types of animals serving astemplates.

FIG. 16 is an electrophoresis photograph showing the results of thedetection of DNA, using mammal-specific primer pairs, using variousprimer pairs, in mixed feed for livestock that contains cattle meat andbonemeal.

FIG. 17 is an electrophoresis photograph showing the results of thedetection of DNA, using ruminant-specific primer pairs, in mixed feedfor livestock that includes cattle meat and bonemeal.

FIG. 18 is an electrophoresis photograph showing the results of thedetection of DNA, using cattle-specific primer pairs, in mixed feed forlivestock that contains cattle meat and bonemeal.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The mitochondrial genome is essential for the biosynthesis of theenzymes required for oxidative phosphorylation (electron transportsystem), and mitochondrial DNA codes for ATP synthase and cytochrome coxidase, and the like. ATP synthase is composed of several subunits, andas discussed above, the inventors found that the ATP synthase subunit 8gene (atp8 gene) is present in the animal mitochondrial genome buthomologous sequence with animal atp8 gene is not present in the plant(Oryza sativa) mitochondrial genome. It thus became possible to identifyanimal species even when plant-derived DNA is present in largequantities in the feed.

Several animal atp8 genes are known. For example, taking the cattle atp8DNA sequence (cattle) as a reference, the atp8 DNA sequences of severalanimal species were aligned so that homology with the cow sequence isachieved, and this is shown in FIG. 1. FIG. 1 shows the DNA sequence ofthe atp8 gene of 1: cattle, 2: alpaca, 3: cat, 4: dog, 5: goat (1), 6:goat (2), 7: horse, 8: antelope, 9: mouse, 10: rabbit, 11: rat, 12:donkey, 13: sheep, 14: deer, 15: sperm whale, and 16: razorback whale.It should be noted that the arrow in the diagram indicates the startposition of the cattle atp8 reading frame.

FIG. 2 shows the atp8 genes of cattle, chicken, and pig aligned with oneanother. Further, FIG. 3 shows the atp8 genes of fish, chicken, cattle,sheep, and pig aligned with one another, and the underlined portionindicates the position of the atp8 gene of a pilchard.

As shown in FIGS. 1 to 3, there is diversity of DNA sequences among theatp8 genes. This diversity can be used to detect specific sequences invarious types of animals, thereby allowing various animal species to beidentified.

To identify various types of animals, examples of methods for detectinga DNA sequence specific to an animal species include Southern Blottingand PCR (polymerase chain reaction). PCR is preferably used in thepresent invention because it permits detection even with very small DNAsamples and it allows accuracy to be improved. In the present invention,PCR is carried out using a pair of primers including a DNA sequencespecific to a target animal species, and a DNA fragment that isamplified is detected.

PCR is for example carried out as follows. First, two regions areselected as primers from any of regions of the atp8 gene and regions,proximal thereto having a DNA sequence specific to a target animalspecies, and the selected primers are synthesized. Using this pair ofprimers, the DNA fragment of the region sandwiched between the twoprimers is amplified by PCR. A sample containing the amplified DNAfragments is then subjected to electrophoresis to determine whether thatDNA fragment is present.

A primer is a DNA sequence of nucleotides in any length, and is suitablyselected based on the sequence alignment of the atp8 genes of varioustypes of animals in publicly available databases. For example, in orderto detect DNA specific to mammals, a region having higher homology withmammals and low homology with non-mammals and having appropriatenucleotide at the 3′ end can be chosen. In order to detect DNA specificto a certain animal, a region having specificity to that animal and lowspecificity with other animals can be chosen. Since primers are used inpairs, two regions, that is, a region on the 5′ side and a region on the3′ side, are selected. If more than two regions can be selected asprimers, then it is possible to use various primer combinations.

For example, FIG. 4 shows a DNA sequence of cattle mitochondrial atp8gene, and the markings in the diagram are the regions (sequences) thatcan be used as primers for detecting cattle-specific DNA. DNA sequencesfor specifically detecting mammals (anicon5 [SEQ ID NO: 1] and anicon3[SEQ ID NO: 2]) and DNA sequences for specifically detecting ruminants(rumicon5 [SEQ ID NO: 3] and rumicon3 [SEQ ID NO: 4]) are also shown inFIG. 4. A more detailed explanation follows in the Examples. Forexample, mammal specific DNA can be detected when anicon5 is used as the5′ primer and anicon3 is used as the 3′ primer. Likewise, ruminantspecific DNA can be detected when rumicon5 and rumicon3 are used asprimers. Alternatively, although not shown, it is also possible tocombine Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6] to detect ruminantspecific DNA with greater accuracy. As the 5′ primer, any one of cow5[SEQ ID NO: 7], cow51 [SEQ ID NO: 8], cow52 [SEQ ID NO: 9], cow53 [SEQID NO: 10], and cow54 [SEQ ID NO: 11] is used, and as the 3′ primer,either cow3 [SEQ ID NO: 12] or cow31 [SEQ ID NO: 13] is used. Forexample, the primer pairs of cow52 and cow31, cow52 and cow3, cow54 andcow31, cow53 and cow3, cow54 and cow3, and cow51 and cow3 are capable ofdetecting cattle-specific DNA. Alternatively, although not shown, it isalso possible to combine Fpc-F [SEQ ID NO: 14] and Fpc-R [SEQ ID NO: 15]to detect cattle-specific DNA with greater accuracy.

The regions surrounded by squares in FIGS. 1 to 3 indicate examples ofDNA sequences used in tests for specifically detecting DNAs derived fromthe respective animal species.

The DNA sequences of the regions selected as primers are synthesized bynormally employed methods. Typically, nucleotides are extended on asupport medium using an automated DNA synthesizer, then removed fromprotecting group and cleaved from the support medium. Then, they can bepurified using a normally employed method (such as columnchromatography) to obtain primers of interest.

Samples that can be measured include raw meat, raw fish, processed meatfood products, processed fish food products, food products containingprocessed meat, food products containing processed fish, blood, hair,body fluids, milk, milk processing products, meat and bonemeal,bonemeal, fishmeal, fish soluble, and feed, fertilizer, and feedadditive containing them. Extraction of mitochondrial DNA from thesesamples is, for example, performed as follows. Approximately 50 mg to500 mg of a sample (for example, 50 mg in the case of raw meat, 100 mgto 500 mg in the case of a dried powder sample) is suspended in about 10times that amount of buffered solution, ground using a bead grindingmethod, for example, and then extracted using a commercially availabletissue cell mitochondrial DNA extraction kit (manufactured by Wako PureChemical Industries, Ltd., for example). Such kits allow purermitochondrial DNA to be collected in that little genome DNA in thetissue cells is contaminated. For example, to the sample ground is addeda reagent in the kit, and centrifuged, and the pellets are collected toconcentrate. It is thus possible to more efficiently extractmitochondrial DNA with little contamination of genome DNA, which ispresent in large quantities in the sample. The method for extracting DNAis not limited to those described herein, and it is apparent to thoseskilled in the art to employ other methods for extracting DNA.

There are no particular limitations regarding the amount of primer used,but generally is it preferable that approximately 0.4 μM is used.

PCR is performed on the pretreated sample, that is, either themitochondrial DNA or total DNA extracted from the sample, using theprimer pair selected above, so as to amplify the DNA fragment of theregion sandwiched between the primers. PCR is executed under conditionsin which it is ordinarily performed, and conditions that are appropriatefor each primer pair are set. For example, DNA fragment is, firstly,heat denatured at 95° C. for 9 minutes; then subjected to the cycle ofreactions of denaturing at 92° C. for 30 seconds to one minute,annealing at 40 to 65° C. for 30 seconds to two minutes, and extendingat 72° C. for 30 seconds to two minutes, which is repeated 30 to 50times; and finally, allowed to react at 72° C. for five minutes tofinish PCR. AmpliTaq GOLD polymerase or the like is used as the DNApolymerase. The size of the PCR product (DNA fragment) amplified by theprimer pair can be approximately 100 bp to approximately 300 bp,although this varies depending on the number of bases between the primerpair that has been selected. The PCR product is then subjected toagarose or polyacrylamide gel electrophoresis, for example, underconditions in which the above DNA fragments can be separated.

The DNA fragments on the gel subjected to electrophoresis can bedetected by detection means normally employed by those skilled in theart, such as ethidium bromide staining, silver staining, fluorescencedetection, and Southern hybridization, and can be confirmed by DNAsequencing.

Thus, if mitochondrial DNA derived from a species of interest is presentin the sample, then, amplified DNA fragments can be detected on the gel.Limits to detection may vary depending on various factors, such as thetype and combination of the primer pair used, the amount of sample, thePCR conditions, and the detection method. If appropriate conditions areselected, then the presence of DNA can be detected with hid sensitivityeven in trace samples. For example, if appropriate conditions areselected when the sample is a mixed feed that contains cattle-derivedmeat and bonemeal, then contamination of only 0.001 wt % cattle meat andbonemeal in the feed can be detected using a specific cattle-specificprimer pair.

It should be noted that when a sample is a mixed feed, if a plantDNA-specific primer pair (for example, the combination of placon5 [SEQID NO: 42] and placon3 [SEQ ID NO: 43]) is used as a control experiment,then it is possible to confirm whether or not DNA has been appropriatelyextracted from the sample. Furthermore, by using a plant DNA-specificprimer pair, a plant-derived component present in a sample can bedetected.

The primer pairs of the present invention may be provided in the form ofa kit for detecting an animal-derived component present in a sample. Thekit comprises at least one of primer pair specific for any animaldescribed above. Preferably, the kit further comprises the plantDNA-specific primer pair as a control.

It should be noted that the primer sequence has been specified in theExamples discussed below, but there is no intention to limit the presentinvention to the following Examples. It is intended that the presentinvention includes in its scope sequences that include those primersequences, or those sequences with one or more base sequencesubstitutions, deletions, or insertions or addition of nucleotidesequences to the 5′ end, and sequences which by changing the hybridizingconditions can hybridize with a DNA of interest and allow a DNA derivedfrom a specific animal species to be detected specifically.

EXAMPLES Primer Synthesis

Primer regions to be used in the following Examples were selected basedon the sequence alignment of the atp8 gene and regions proximal theretoof various animals shown in FIGS. 1 to 3. The DNA sequences of theselected regions were synthesized using an automated DNA synthesizer.

Example 1 Specific Detection of Mammal-Derived DNA Sequence

DNAs from 15 types of meat samples, these being cattle, sheep, goat,deer, pig, horse, rabbit, whale, chicken, codfish, salmon, sardine,crab, prawn, and clam, were prepared as follows. Each DNA sample wasdiluted in an appropriate concentration in buffer solution (10 mMTris-HCl, pH 7.5, 20 mM EDTA, pH 7.5), ground using a bead grindingmethod, and then DNA was extracted using the mtDNA Extractor CT Kit(manufactured by Wako Pure Chemical Industries, Ltd.), which is acommercially available tissue cell mitochondrial DNA extraction kit.

With each of these DNA samples serving as a template, PCR was performedusing anicon5 [SEQ ID NO: 1] and anicon3 [SEQ ID NO: 2] as the 5′ primerand the 3′ primer, respectively. The PCR conditions were as follows:reaction buffer solution (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mMMgCl₂, 0.001% (w/v) gelatin); heat denaturing at 95° C. for 9 minutes,followed by the cycle of reactions of denaturing at 92° C. for oneminute, annealing at 55° C. for two minutes, and extending for twominutes at 72° C. repeated 45 times, and lastly, the product was allowedto react at 72° C. for five minutes. After PCR, the reaction mixture wassubjected to agarose gel electrophoresis, and the PCR product (DNAfragments) was detected by ethidium bromide staining. The results areshown in FIG. 5. In the diagram, M is the molecular weight marker.

As is clear from FIG. 5, by performing PCR using anicon5 [SEQ ID NO: 1]and anicon3 [SEQ ID NO: 2] as primers with each animal-derived DNAserving as a template, the PCR products (amplified DNA fragments) wereobserved only in mammals (176 bp band position) (lanes 1 to 8). That is,it was demonstrated that by using anicon5 [SEQ ID NO: 1] and anicon3[SEQ ID NO: 2] as the primer pair, it is possible to specifically detectonly mammal-derived DNA from among various types of animal meat samples.Consequently, the combination of anicon5 [SEQ ID NO: 1] and anicon3 [SEQID NO: 2] is a primer pair that specifically detects the DNA sequencederived from animal species of mammalian origin. The regions that can beused as primers for detecting DNAs derived from mammals are shown inFIG. 4. It should be noted that this primer pair was designed so as tofail to amplify human DNA.

Example 2 Specific Detection of Ruminant-Derived DNA Sequence-1

With the 15 types of DNA samples prepared in the same manner as inExample 1 (except that a different type of cattle DNA sample was used inplace of deer) serving as templates, PCR was performed using rumicon5[SEQ ID NO: 3] and rumicon3 [SEQ ID NO: 4] as the 5′ primer and the 3′primer, respectively. The PCR conditions were as follows: heatdenaturing at 95° C. for 9 minutes, followed by the cycle of reactionsof denaturing at 92° C. for one minute, annealing at 45° C. for twominutes, and extending at 72° C. for two minutes repeated 45 times, andlastly, the product was allowed to react at 72° C. for five minutes.After PCR, the reaction mixture was subjected to agarose gelelectrophoresis, and the PCR product (DNA fragments) was detected byethidium bromide staining. The results are shown in FIG. 6.

As shown in FIG. 6, by performing PCR using rumicon5 [SEQ ID NO: 3] andrumicon3 [SEQ ID NO: 4] as primers with each animal-derived DNA servingas a template, PCR products from DNAs derived from beef cattle, dairycattle, sheep, and goat, which are ruminants, were observed (bandposition indicated by the arrow). On the other hand, PCR products (DNAfragments) could not be detected in the DNA samples of animals that werenot ruminants. That is, it was demonstrated that by using rumicon5 [SEQID NO: 3] and rumicon3 [SEQ ID NO: 4] as the primer pair, it is possibleto specifically detect only ruminant-derived DNA from various types ofanimal meat samples. Consequently, the combination of rumicon5 [SEQ IDNO: 3] and rumicon3 [SEQ ID NO: 4] is a primer pair that specificallydetects the DNA sequence derived from animal species of ruminant origin.The regions that can be used as the primers for detecting DNAs derivedfrom ruminants are shown in FIG. 4.

It should be noted that in rumicon5 [SEQ ID NO: 3], “r” is “g” or “a”,and “k” is “g” or “t”. That is, rumicon5 [SEQ ID NO: 3] is a primermixture, but it should be apparent to those skilled in the art that eachcan be used independently.

Example 3 Specific Detection of Ruminant-Derived DNA Sequence-2

With the 15 types of DNA samples prepared in the same manner as inExample 1 serving as templates, PCR was performed using Fpr-F [SEQ IDNO: 5] and Fpr-R [SEQ ID NO: 6] as the 5′ primer and the 3′ primer,respectively. The PCR conditions were as follows: heat denaturing at 95°C. for 9 minutes, followed by the cycle of reactions of denaturing at92° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extendingat 72° C. for 30 seconds repeated 45 times, and lastly, the product wasallowed to react at 72° C. for five minutes. After PCR, the reactionmixture was subjected to agarose gel electrophoresis, and the PCRproduct (DNA fragments) was detected by ethidium bromide staining. Theresults are shown in FIG. 7.

As shown in FIG. 7, by performing PCR using Fpr-F [SEQ ID NO: 5] andFpr-R [SEQ ID NO: 6] as primers with each animal-derived DNA serving asa template, PCR products from DNAs derived from cattle, sheep, goat, anddeer which are ruminants, were more clearly observed (104 bp bandposition). On the other hand, PCR products (DNA fragments) could not bedetected in the DNA samples of animals that were not ruminants. That is,it was demonstrated that by using Fpr-F and Fpr-R as the primer pair, itis possible to more specifically detect only ruminant-derived DNA fromvarious types of animal meat samples. Consequently, the combination ofFpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6] is a primer pair thatspecifically detects DNAs derived from animal species of ruminantorigin.

It should be noted that in Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO:6], “r” is “g” or “a”, and “k” is “g” or “t”. That is, these are primermixtures, but it should be apparent to those skilled in the art thateach can be used independently.

Example 4 Specific Detection of Cattle-Derived DNA Sequence-1

Specific detection of a cattle-derived DNA sequence was performed usingthe various primer pairs listed in Table 1. PCR was performed under thesame conditions as those of Example 1, except that DNAs prepared fromcattle, sheep, goat, pig, and chicken meat were served as templates. Theresults are shown in FIGS. 8 to 10.

TABLE 1 Primer Anealing Result 5′ cow51 SEQ ID NO: 8 55° C. 2 min FIG. 83′ cow3 SEQ ID NO: 12 5′ cow52 SEQ ID NO: 9 52° C. 2 min FIG. 9 3′ cow3SEQ ID NO: 12 5′ cow53 SEQ ID NO: 10 52° C. 2 min FIG. 9 3′ cow3 SEQ IDNO: 12 5′ cow54 SEQ ID NO: 11 52° C. 2 min FIG. 9 3′ cow3 SEQ ID NO: 125′ cow5 SEQ ID NO: 7 52° C. 2 min FIG. 9 3′ cow31 SEQ ID NO: 13 5′ cow51SEQ ID NO: 8 56° C. 2 min FIG. 10 3′ cow31 SEQ ID NO: 13 5′ cow52 SEQ IDNO: 9 52° C. 2 min FIG. 10 3′ cow31 SEQ ID NO: 13 5′ cow53 SEQ ID NO: 1056° C. 2 min FIG. 10 3′ cow31 SEQ ID NO: 13 5′ cow54 SEQ ID NO: 11 56°C. 2 min FIG. 10 3′ cow31 SEQ ID NO: 13 5′ cow5 SEQ ID NO: 7 46° C. 2min not shown 3′ cow3 SEQ ID NO: 12

The results shown in FIG. 8 indicate that if cow51 [SEQ ID NO: 8] andcow3 [SEQ ID NO: 12] are used as the primer pair, then DNA fragments thesame size as the PCR product (DNA fragments) obtained fromcattle-derived meat did not find when sheep, goat, pig, or chicken DNAserved as templates. It is clear that the combination of cow52 and cow3of FIG. 9 and the combination of cow52 and cow31 of FIG. 10 areparticularly suitable primer pairs for cattle-specific DNA detection,because similar DNA fragments were not detected in the samples of otheranimal species. On the other hand, as shown in FIG. 9, when cow5 [SEQ IDNO: 7] and cow31 [SEQ ID NO: 13] are used as the primer pair, a band ofthe same size as the DNA fragment obtained using a cattle DNA as atemplate was slightly found in sheep. Likewise, in FIG. 10, when theprimer pair cow51 [SEQ ID NO: 8] and cow31 [SEQ ID NO: 13] and theprimer pair cow53 [SEQ ID NO: 10] and cow31 [SEQ ID NO: 13] are used, aband the same size as the DNA fragment obtained using cattle DNA as atemplate was observed.

Example 5 Specific Cattle-Derived DNA Sequence-2

With the 15 types of DNA samples prepared in the same manner as inExample 1 serving as templates, PCR was performed using Fpc-F [SEQ IDNO: 14] and Fpc-R [SEQ ID NO: 15] as the 5′ primer and the 3′ primer,respectively. The PCR conditions were as follows: heat denaturing at 95°C. for 9 minutes, followed by the cycle of reactions of denaturing at92° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extendingat 72° C. for 30 seconds repeated 45 times, and lastly, the product wasallowed to react at 72° C. for five minutes. After PCR, the reactionmixture was subjected to agarose gel electrophoresis, and the PCRproduct (DNA fragments) was detected by ethidium bromide staining. Theresults are shown in FIG. 11.

As shown hi FIG. 11, by performing PCR using Fpc-F [SEQ ID NO: 14] andFpc-R [SEQ ID NO: 15] as primers with each animal-derived DNA serving asa template, the PCR product from only cattle-derived DNA was detected(126 bp band position). That is, it was demonstrated that by using Fpc-Fand Fpc-R as the primer pair, it is possible to detect with extremeSpecificity only cattle-derived DNA from various (types of animal meatsamples. Consequently, the combination of Fpc-F [SEQ ID NO: 14] andFpc-R [SEQ ID NO: 15] is a primer pair that specifically detects DNAsderived from animal species of bovine origin.

Example 6 Specific Detection of Pig-Derived DNA Sequence-1

Specific detection of a pig-derived DNA sequence was performed using thevarious primer pairs listed in Table 2. PCR was performed under the sameconditions as those of Example 1, except that DNAs prepared from cattle,sheep, goat, pig, and chicken meat were served as templates.

TABLE 2 Primer Anealing Result 5′ pig5 SEQ ID NO: 16 46° C. 2 min notshown 3′ pig3 SEQ ID NO: 19 5′ pig51 SEQ ID NO: 17 58° C. 2 min FIG. 123′ pig3 SEQ ID NO: 19 5′ pig5 SEQ ID NO: 16 46° C. 2 min not shown 3′pig31 SEQ ID NO: 20 5′ pig5 SEQ ID NO: 16 46° C. 2 min not shown 3′pig32 SEQ ID NO: 21 5′ pig51 SEQ ID NO: 17 55° C. 2 min not shown 3′pig31 SEQ ID NO: 20 5′ pig51 SEQ ID NO: 17 55° C. 2 min not shown 3′pig32 SEQ ID NO: 21

FIG. 12 shows the results obtained by using pig51 [SEQ ID NO: 17] andpig3 [SEQ ID NO: 19] as the primer pair. As is clear from FIG. 12, PCRproducts (DNA fragments) were detected only in the sample derived frompig meat (band position indicated by the arrow). Consequently, thisprimer pair is useful for specifically detecting pig-derived DNAsequences.

Example 7 Specific Detection of Pig-Derived DNA Sequence-2

With the 15 types of DNA samples prepared in the same manner as inExample 1 serving as templates, PCR was performed using pig5-3 [SEQ IDNO: 18] and pig32-2 [SEQ ID NO: 22] as the 5′ primer and the 3′ primer,respectively. The PCR conditions were as follows: heat denaturing at 95°C. for 9 minutes, followed by the cycle of reactions of denaturing at92° C. for 30 seconds, annealing at 60° C. for 1 minute, and extendingat 72° C. for 1 minute repeated 45 times, and lastly, the product wasallowed to react at 72° C. for five minutes. After PCR, the reactionmixture was subjected to agarose gel electrophoresis, and the PCRproduct (DNA fragments) was detected by ethidium bromide staining. Theresults are shown in FIG. 13.

As shown in FIG. 13, by performing PCR using pig5-3 [SEQ NO: 18] andpig32-2 [SEQ ID NO: 22] as primers with each animal-derived DNA servingas a template, the PCR product from only DNA derived from pig wasobserved (band position indicated by the arrow). On the other hand, PCRproducts could not be detected in the DNA samples of other animals. Thatis, it was demonstrated that by using pig5-3 and pig32-2 as the primerpair, it is possible to detect with only pig-derived DNA among varioustypes of animal meat samples. Consequently, the combination of pig5-3[SEQ ID NO: 18] and pig32-2 [SEQ ID NO: 22] is a primer pair that veryspecifically detects DNAs derived from animal species of pig origin.

Example 8 Specific Detection of Sheep-Derived DNA Sequence

Specific detection of a sheep-derived DNA sequence was performed usingsheep5 [SEQ ID NO: 23] and sheep3 [SEQ ID NO: 24] as the 5′ primer andthe 3′ primer, respectively. PCR was performed under the same conditionsas those of Example 1, except that DNAs prepared from cattle, sheep,goat, pig, and chicken meat were served as templates and that theannealing conditions were set to two minutes at 46° C. The results areshown in FIG. 14.

These results demonstrate that specific detection of a sheep-derived DNAsequence is possible using sheep5 [SEQ ID NO: 23] and sheep3 [SEQ ID NO:24] as the primer pair.

Example 9 Specific Detection of Goat-Derived DNA Sequence

Specific detection of a goat-derived DNA sequence was performed usinggoat5 [SEQ ID NO: 25] and goat3 [SEQ ID NO: 26] as the 5′ primer and the3′ primer, respectively. PCR was performed under the same conditions asthose of Example 1, except that DNAs prepared from cattle, sheep, goat,pig, and chicken meat were served as templates and that the annealingconditions were set to two minutes at 46° C. The results are shown inFIG. 14.

These results demonstrate that specific detection of a goat-derived DNAsequence is possible using goat5 [SEQ ID NO: 25] and goat3 [SEQ ID NO:26] as the primer pair.

Example 10 Specific Detection of Chicken-Derived DNA Sequence

Specific detection of a chicken-derived DNA sequence was performed usingthe various primer pairs listed in Table 3 as the 5′ and the 3′ primers.PCR was performed under the same conditions as those of Example 1,except that DNAs prepared from cattle, sheep, goat, pig, and chickenmeat were served as templates. The results obtained by using chick5 [SEQID NO: 28] and chick3 [SEQ ID NO: 30] as the primers are shown in FIG.15.

TABLE 3 Primer Anealing Result 5′ chick5 SEQ ID NO: 28 46° C. 2 min FIG.15 3′ chick3 SEQ ID NO: 30 5′ chick51 SEQ ID NO: 29 50° C. 2 min notshown 3′ chick3 SEQ ID NO: 30 5′ chick5 SEQ ID NO: 28 50° C. 2 min notshown 3′ chick31 SEQ ID NO: 31 5′ chick51 SEQ ID NO: 29 60° C. 2 min notshown 3′ chick31 SEQ ID NO: 31

As shown in FIG. 15, specific detection of a chicken-derived DNAsequence is possible using Chick5 [SEQ II) NO: 28] and chick3 [SEQ IDNO: 30] as the primer pair (band position indicated by the arrow).

Example 11 Detection of Cattle-Derived Component in Mixed Feed-1

Meat, and bonemeal derived from cattle (Australian bred) was mixed intomixed feed for livestock (main components: corn, milo, gluten feed,bran, rice bran, soy oil cake, rapeseed oil cake) at a predeterminedratio. Then, as in Example 1, 100 mg of the feed obtained was suspendedin ten times that amount of buffer solution, and ground by a beadgrinding method. Then, DNA was extracted using a commercially availabletissue cell mitochondrial DNA extraction kit (manufactured by Wako PureChemical Industries, Ltd., Japan). Using this DNA as a template, PCR wasperformed under the same conditions as in Example 1 using anicon5 [SEQID NO: 1] and anicon3 [SEQ ID NO: 2], which are the primer pair forspecifically detecting mammalian-derived DNA sequences used in Example1, Fpr-F [SEQ ID NO: 5] and Fpr-R [SEQ ID NO: 6], which are the primerpair for specifically detecting ruminant-derived DNA sequences used inExample 3, and Fpc-F [SEQ ID NO: 14] and Fpc-R [SEQ ID NO: 15], whichare the primer pair for specifically detecting cattle-derived DNAsequences used in Example 5. The results are shown in FIG. 16. In allcases where these primer pairs were used, it was possible to detectcattle meat and bonemeal contained in the feed at 0.01 wt %.

Example 12 Detection of Cattle-Derived Component in Mixed Feed-2

PCR was performed under the same conditions as in Example 2, except thatDNA extracted from mixed feed in the same manner as in Example 11 wasserved as a template and that the primer pair rumicon5 [SEQ ID NO: 3]and rumicon3 [SEQ ID NO: 4] for specifically detecting ruminant-derivedDNA sequences that is used in Example 2 was used. The results are shownin FIG. 17. Detection was possible even in a case where the feedcontained 0.1 to 1 wt % meat and bonemeal.

Example 13 Detection of Cattle-Derived Component in, Mixed Feed-3

PCR was performed under the same conditions as in Example 3, except thatDNA extracted from mixed feed in the same manner as in Example 11 wasserved as a template and that the cattle derived DNA-specific primerpair of cow52 [SEQ ID NO: 9] and cow31 [SEQ ID NO: 13] that was used inExample 3 was used. The results are shown in FIG. 18. Detection waspossible even in a case where meat and bonemeal was only contained inthe feed at a mere 0.001 wt %.

Example 14 Specific Detection of Fish-Derived DNA Sequence

Fishmeal is often a mixture of various types of fish. Thus, severalprimer regions were selected based on the alignment of the DNA sequencesof the atp8 gene and proximal regions shown in FIG. 3 in order to detecta wide range of components derived from various types of fish. Theunderlined portion in FIG. 3 is the atp8 gene of a pilchard, and theasterisk bellowing the alignment indicates the portion where bases areconserved among the DNA sequences.

A commercially available tissue cell mitochondrial DNA extraction kit(manufactured by Wako Pure Chemical Industries, Ltd., Japan) was used inthe same manner as in Example 1 to prepare DNA from the 26 types ofplants and animals shown in Table 4 below. PCR was carried out usingthis DNA solution as a template and using the various combinations of 5′and 3′ primers selected based on the sequence alignment of FIG. 3. Asfor the primers used, the 5′ side primers used were fish5 [SEQ ID NO:32], fish51 [SEQ ID NO: 34], fish5-1 [SEQ ID NO: 38], and fish5-2 [SEQID NO: 39], and the 3′ side primers used were fish3-1 [SEQ ID NO: 33],fish3 [SEQ ID NO: 35], fish31 [SEQ ID NO: 36], fish31-1 [SEQ ID NO: 37],fish3-2 [SEQ ID NO: 40], and fish3-3 [SEQ ID NO: 41]. The PCR conditionswere as follows: reaction buffer solution (10 mM Tris-HCl, pH 8.3, 50 mMKCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin); heat denaturing at 95° C. for9 minutes, followed by the cycle of reactions of denaturing at 92° C.for 30 seconds, annealing at 55° C. for 30 seconds, and extending at 72°C. for 30 seconds repeated 45 times, and lastly, the product was allowedto react at 72° C. for five minutes. After PCR, the reaction mixture wassubjected to agarose gel electrophoresis, and the PCR product (DNAfragments) was detected by ethidium bromide staining. In the positivecontrol, PCR was performed with a solution of sardine-derived DNAobtained in the same manner as in Example 1 from crushed sardine meatserving as the template. In the negative control, PCR was performed witha blank in which the template DNA was not added to the PCR reactionmixture.

Table 4 shows the results obtained with the combination of fish5 [SEQ IDNO: 32] and fish3-1 [SEQ ID NO: 33]. It should be noted that in theassessment column of Table 4, the cases where a band was not detected inthe negative control, but a band of a target size was detected in thepositive control, and a band of the same size as that detected in thepositive control was detected in the sample, were regarded as positive.

TABLE 4 Animal Assesment* Cattle(Beef cattle) − Cattle(Holstein) −Cattle(Japanese black) − Cattle(Red hair) − Cattle(F2) − Sheep − Goat −Deer − Pig − Horse − Rabbit − Whale − Chicken − Human − Scad − Sardine +Flatfish ± Salmon + Alaska pollack + Tuna + Crab − Lady crab + Prawn −Squid − Clam − Corn − *+; Detection of band at 284 bp, ±; Detection ofunclear band at 284 bp, −; Under detection limit of band at 284 bpDetection was performed with agarose gel electrophoresis and ethidiumbromide staining.

As shown in the assessment column of Table 4, a band was detected at theposition of 284 bp in DNA samples taken from sardine, flatfish, salmon,Alaska Pollack, tuna, and lady crab. On the other hand, a band was underdetectable in mammalian, chicken, and corn DNA samples. Consequently, itis clear that the primer pair of fish5 [SEQ ID NO: 32] and fish34 [SEQID NO: 33] allows the major fish types often used to produce fishmeal tobe detected.

Apart from this, about 100 kinds of fishmeal was examined by extractingDNA in the same manner as above. The results showed that a band can bedetected at the position of interest in about 90 percent of thefishmeal. These results also indicated that wide ranging detection ispossible for DNA derived from various types of fishes.

Example 15 Fish-Derived DNA Sequence Addition Testing and DetectionLimit

Whether or not it is possible to detect fishmeal mixed into commerciallyavailable mixed feed for raising cattle or chickenmeal was examined. Theraw material of the fishmeal was mackerel and saury, and the fishmealused was passed through a 0.5 mm mesh wire sieve. As regards the mixedfeed, commercially available mixed feed for cattle was crushed with acrusher and a triturator, and was passed through a 0.5 mm mesh wiresieve. It should be noted that the mixture proportions of the mixed feedare as shown below in Table 5. The chickenmeal was commerciallyavailable chickenmeal, and had been passed through a 0.5 mm mesh wiresieve.

TABLE 5 Type of Ratio Feed material (%) Raw materials Mixed feed forChaff and bran 37 Corn gluten feed, Rice bran, raising dairy Bran,Soybean hull cattle Cereals 36 Corn, Rye, Wheat flour, Milo Vegetableoil 19 Rapeseed oil cake, Soy oil cake cake Others 8 Syrup, Calciumcarbonate, Salt Mixed feed for Cereals 66 Corn, Barley, Wheat raisingbeef Chaff and bran 28 Bran, Corn gluten feed cattle Vegetable oil 4 Soyoil cake cake Others 2 Alfalfa meal

Samples containing 1 wt % fishmeal in the above mixed feed andchickenmeal were prepared. As regards these samples and the sampleswithout fishmeal, DNAs were extracted as in Example 1, and PCR wasperformed using the primer pair of fish5 [SEQ ID NO: 32] and fish3-1[SEQ ID NO: 33].

Fish-derived DNA was detected in the mixed feed and chickenmealcontaining 1 wt % fishmeal. On the other hand, DNA was not detected fromthe samples to which fishmeal was not added. Consequently, it was clearthat the fishmeal used in this example can be detected even whencontained at 1 wt %.

Next, the detection limit of fishmeal contaminated in mixed feed wasexamined. Fishmeal was added at 10, 1, 0.1, and 0.01 wt % to the mixedfeed for raising dairy cattle of Table 5, and using the same procedureas that of Example 14, DNA was extracted in duplicate for each sample,and for each extracted sample PCR was performed in duplicate. When bothof the two DNA extracts resulted in positive, an assessment of positivewas reached. The detection limit for the fishmeal when using the primerpair of fish5 [SEQ ID NO: 32] and fish3-1 [SEQ ID NO: 33] was about 1 wt%.

The invention may be embodied in other forms without departing from thespirit or essential characteristics thereof. The embodiments disclosedin this application are to be considered in all respects as illustrativeand not limiting. The scope of the invention is indicated by theappended claims rather than by the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

What is claimed is:
 1. A method for detecting a mammal-specific DNAusing a primer pair, wherein the primer pair is a combination of the DNAsequence ID NO: 1 and the DNA sequence of SEQ ID NO:
 2. 2. The methodaccording to claim 1, wherein a mammal of the mammal-specific DNA isselected from the group consisting of cattle, sheep, goat, deer, pig,horse, rabbit, and whale.
 3. A kit for detecting an animal-derivedcomponent present in a sample comprising: at least one primer pair of acombination of the DNA sequence ID NO: 1 and the DNA sequence of SEQ IDNO:
 2. 4. The kit according to claim 3, wherein an animal of theanimal-derived component is selected from the group consisting ofcattle, sheep, goat, deer, pig, horse, rabbit, and whale.